The invention relates to the field of polymer coating of functional groups made by chemical vapor deposition (CVD) and the use of homogenously distributed functional groups for defined surface design.
Methods for isotropic surface modification of materials are well known, especially plasma aching and plasma polymerization (see Yasuda or EP 0519087 A1). Other widely used methods include laser treatment, ion beam treatment or wet chemical modifications. Another technique is based on the adsorption of self-assembled monolayers, which commonly utilizes substrates made of silicon, silicon dioxide, silver, copper or gold.
The research of thiol monolayers on gold surfaces has resulted in brake-drawing technologies such as soft-lithography. Applications of self-assembled monolayers (SAM's) include sensor development, corrosion protection and heterogeneous catalysis and reference surfaces for biocompatibility studies. SAM's have also been used as templates for organic synthesis and layer-by-layer adsorption. All these techniques are based on a common approach: Spontaneous monolayer formation of thiols on gold to achieve a densely packed two-dimensional crystalline structure offers reactive head groups for further modification.
Rather than pure surface modification, for some applications, surface coating is the method of choice. Surface coating methods include carbon like diamond coatings (CLD), carbon nitride coating, deposition of several metal layers or simple spin, dip, or spray coating of polymers. CVD polymerization coatings of paracyclophane or chlorine derivatives thereof, applied in order to achieve inert surfaces (Swarc, Gorham, Union Carbide) have excellent homogeneity, adhesion and stability. Recently CVD coating of functionalized paracyclophanes has been used in order to immobilize bioactive proteins (Lahann Biomaterials 2001, Höcker, DE 19604173 A1).
This coating procedure developed to be a one-step coating and functionalization method offers a wide range of applications since good bulk properties of a material has been maintained combined with enhanced contact properties. The ‘activation’ of surfaces with bivalent spacer molecules offers the opportunity of further modification such as drug immobilization. By using the interfaces for immobilization of proteins, cell receptors, cytokines, inhibitors etc., bioactive surfaces that interact with the biological environment in a defined and active matter can be achieved. The activation of the functional groups of the CVD coating with bivalent spacer molecules requires wet chemical procedures and is connected to the use of solvents and harmful monomer molecules. The reaction steps are not sterile and offer a high risk of contamination of the device. While requiring intensive cleaning procedures, again connected to the use of solvents.
The requirement of an additional activation step therefore annuls some of the main advantages of the CVD coating procedure, like ultra-clean coating conditions resulting from the gas phase process without any solvents or additives. In the same manner, the high biocompatibility resulting from those features may be drastically decreased.
CVD-based polymer coatings are used in order to provide amino- or hydroxyl-functionalized surfaces for conjugation of biomolecules. Amino- or hydroxyl-functionalized poly(para-xylylene) coatings however still require an additional activation step for linkage of proteins or ligands. Typically, bivalent spacers such as hexamethylene diisocyanate have been used for amino- or hydroxyl-functionalized polymers. The additional activation step not only limits the feasibility of microengineering, but also causes the contamination of the substrate with organic solvents and volatile chemicals. The contamination reduces crucial advantages of CVD coatings, such as low intrinsic cytotoxicity due to the lack of harmful solvents, initiators, or accelerators during polymerization. Therefore, a one-step coating procedure that provides linkable reactive groups is highly desirable.
The control of engineered microenvironments on device surfaces has been addressed by several approaches including soft lithographic methods such as microcontact printing (μCP) and micromolding (MIMIC). These procedures have been used for the formation of a wide range of surface patterns, e.g. protein and cell arrays and for micro- and nanofabrication of devices. Potential applications include the regulation of cell shapes, the development of microelectronic elements such as optical displays, circuits, or lasers and the fabrication of complex three-dimensional microstructures or microfluidic devices. One of the most important steps is the spatially controlled self-assembly of monolayers on a substrate. A number of prior art systems have been investigated, however only assemblies of siloxanes on silicon oxide and of alkanethiolates on gold have been widely exploited.
Biomedical devices are typically manufactured from polymers and metals other than gold. Microengineering of patterns is very challenging. The main limitation is the lack of sufficient and homogeneously distributed functional groups on the substrate surface, which are necessary for the build-up of further structural elements. Treatment with high-energy sources such as plasma, laser, or ion beams has been used to create functionalized surfaces for biomedical systems. Poly(ethylene terephthalate) is surface-modified via multi-step synthesis to generate a surface for μCP of biological ligands. Patterns of proteins or cells have been also generated by means of photolithographic techniques. Examples include the spatially controlled photoablation of previously adsorbed proteins and the linkage of proteins via photosensitive groups. Photolithography however tends to be laborious and expensive. Over the past few years, the combination of chemisorption of alkanethiolates onto gold surfaces and soft lithographic methods has been shown to be a versatile technique for fabrication of patterned surfaces. Among soft lithographic methods, microcontact printing (μCP) of alkanethiolates to gold or silver substrates has been most intensively used for generating patterns of various mammalian cells. Although immensely important for a broader fundamental understanding of cell shape, position and function, the biomedical applications of these techniques are limited by relevant devices being mostly manufactured out of materials other than gold or silver.
The predictable design of a surface with different domains of functionality is a much stronger approach, since it opens the entrance to microstructured surfaces. It combines the chemical flexibility of adsorption-based techniques such as SAM on gold with the mechanical stability of a robust CVD-polymer coating. Generally, a generic method to fabricate patterns of proteins and/or cells based on reactive coating and spatially directed self-assembly includes generating a pattern of endothelial cells via a three-step procedure. First, a substrate is coated by a polymer presenting chemically reactive pentafluorophenol ester groups (reactive coating). Second, patterns are created by microcontact printing (μCP) of amino-derived biotin ligands. Streptavidin is subsequently bound to the biotin-exposing surface regions and served as a linker providing free biotin-binding sites, which are used to bind a biotin-tethered antibody against α-integrin. The specific interaction of the antibody with α-integrin located on the surface of endothelial cells is used for the spatially controlled deposition of cells.
The ability to generate patterns of biological ligands, proteins or cells on surfaces is important for several technologies in biomedical engineering such as the development of certain types of biosensors or fundamental studies of cell biology. The spatially controlled attachment of ligands is also necessary for some biological assays and for combinatorial screening of drugs. In tissue engineering, the formation of tissue or organized cell structures often requires a specific architecture that allows cells to occupy defined locations on a construct, while preventing non-specific adhesion. PCT/US99/15968 discloses arrays of protein-capture agents, which are useful for the simultaneous detection of a plurality of proteins. The arrays comprise a thin organic layer that is between 10 and 20 mm thick. The use of monomolecularly dimensioned interlayers is associated with disadvantages described for self-assembled monolayers. SAM's are restricted to a few substrate materials; porous structures such as foams, scaffolds or membranes are difficult to process and applications in chemically aggressive environments such as in vivo are not possible. The herein disclosed methods allow for overcoming these backdraws.
U.S. Pat. No. 6,103,479 (Taylor) discloses miniaturized cell array methods and apparatus for cell-based screening. These devices can be used with methods of performing high-throughput screening of the physiological response of cells to biologically active compounds and methods of combining high-throughput with high-content spatial information at the cellular and sub-cellular level as well as physiological, biochemical and molecular activities.
Other prior art references which generally describe cell arrays and methods and apparatus to use the same include WO 01/07891 (Kapur et al.), WO 00/60356 (Kapur et al.), U.S. Pat. No. 5,776,748 (Singhvi et al.), and WO 00/53625 (Rossi et al.). However, all of these references include multi-step processes and include the use of solvent.
U.S. Pat. No. 6,192,168 (Feldstein et al.) describes a reflectively coated optical waveguide and fluidics cell integration, which includes a waveguide having a patterned, reflective coating.
Electrophoresis is an indispensable tool of biotechnology as described in PCT Pub. No. WO 99/40174. The devices are used in a variety of applications and preparation of pure samples of nucleic acids, proteins, carbohydrates, the identification of a particular analyte in a complex mixture and the like. They are also used in capillary electrophoresis (CE) and microchannel electrophoresis (MCE). These methods are used for industrial processes and basic research including analytical, biomechanical, pharmaceutical, environmental, molecular, biological, food and clinical applications.
U.S. Pat. Nos. 5,858,188 and 5,935,409 further describe microchannels and their use in electrophoresis and processes for recovering metal values from metal sources containing more than one metal.
Additional references in the field of cell assays include PCT Pub. Nos. WO 00/60356 and WO 01/07891. These disclose methods for making a substrate for selective cell patterning and the substrates themselves; and for array for cell screening and methods of making them, respectively.
Arrays of protein-captive agents are useful for simultaneous detection of a plurality of proteins which are expression products or fragments thereof, of a cell or population of cells as described in PCT WO 00/04389. The arrays are useful for various proteomic applications including assessing patterns of protein expression and modification in cells.